In this paper we present the results of a study of different populations of the genus Tyrrhenoleuctra, which includes morphologically cryptic species, in the Iberian Peninsula. A total of six populations were analysed from a molecular point of view and a behavioural study was conducted on three of them. Two phylogenetic trees, maximum likelihood (ML) and neighbour joining (NJ), were constructed using both new COI sequences and COI sequences already available in GenBank. Furthermore, intra- and interspecific p-distances were calculated within and between sequences of the different populations. As regards behavioural analyses, the male call of individuals from three of the studied populations was recorded, analysed and described. Both phylogenetic approaches used, ML and NJ, were congruent in discriminating five well-defined clusters, representing the four known Iberian Tyrrhenoleuctra species and a previously unknown taxon. The sequences from the six studied populations produced in this study fell within two clades: T. lusohispanica (four populations) and the new taxon (two populations). These results were supported also by the behavioral study, in which the drumming calls of individuals belonging to the two clades differed in some important parameters such as the number of sequences, intersequence interval and interbeat interval. The obtained results expand the previously known distribution of T. lusohispanica and show the existence of a new species (Tyrrhenoleuctra hynesi sp. n.) within this genus from a more restricted area, that will be described in this paper.